Neurons synthesize, sort, and transport numerous membrane proteins for localization to highly specialized functional domains on their plasma membranes. The molecular mechanisms whereby they accomplish this targeting are unknown. The long range goal of this project is to determine the molecular signal(s) responsible for targeting neuronal membrane proteins to their appropriate destinations on the cell surface and the mechanism whereby they are retained within selected regions of the plasma membrane. As a first step in understanding these mechanisms we have chosen to study the processing, transport, and localization of acetylocholinesterase (AChE) in tissue-cultured CNS neurons. AChE is the enzyme that hydrolyses the acetylcholine released by cholinergic nerves. This enzyme exists as a complex family of oligomeric forms, a subset of which are rapidly transported down the axon. In addition, a single integral membrane form of AChE is targeted to the surface plasma membrane in tissue cultured neurons where it is localized in clusters along the neurites. It is likely that the mechanism(s) employed by neurons to sort and transport AChE molecules will be shared by most if not all rapidly transported neuronal membrane proteins, whereas the final targeting will depend upon specific characteristics of subsets of membrane proteins. These are basic studies that will lead to an understanding of how all nerve cells sort and transport membrane proteins to specific regions of their plasma membranes. Our specific aims during the tenure of this proposal are: 1) to determine where, when, and how the signal specifying membrane localization of AChE in CNS neurons occurs; 2) to clone and identify full length cDNAs encoding the AChE polypeptides from chicken CNS neurons using a full length Torpedo AChE cDNA as a probe, 3) to express these cDNAs in mouse L cells to study the fate of the chicken AChE polypeptides, and 4) to inject in vitro synthesized AChE, VSV G-glycoprotein, and IL2 receptor mRNA into identified neurons of the leech (Hirudo medicinalis) to study the transport and localization of an identified rapidly transported protein and foreign membrane proteins in vivo.